The crucial role of homeobox genes in patterning of the vertebrate skeleton is well established. Much is known about the specificity and cooperation of particular Hox genes in defined skeletal regions, but despite more than 10 years of functional experiments in numerous laboratories, it is still unknown how Hox transcription factors actually control vertebral identity. The transcriptional targets in the developing skeleton also have not been identified, a major obstacle to a mechanistic understanding of Hox regulated pathways. Here, I propose a genetic approach to the identification of Hox-controlled pathways; a new direction in the field of skeletal patterning. The central hypothesis is that skeletal patterning-- as regulated by Hox genes--manifests as a quantitative trait, which is controlled by gene-gene and gene-environment interactions. This proposition is supported by extensive data from our laboratory that demonstrate the existence of nutritional and genetic modifiers of Hox gene function in the skeleton. We have shown that (i) genetic factors modulate severity of skeletal anomalies in Hoxb6 knockout mice; (ii) phenotype manifestation is controlled by genetic background of the developing embryo itself; (iii) distinct skeletal elements are affected by region-specific pathways that operate largely independently; and that (iv) the number of genetic modifiers is small, and therefore experimentally tractable. Our major goal now is to define the genetic factors that interact with HoxbG in skeletal patterning by pursuit of the following Specific Aims: (1) to map the genomic location of the strain-specific genetic modifier(s) by microsatellite marker whole genome scan in a backcross of Hoxb6 mutants on C57BI/6 and 129Sv/Ev backgrounds; (2) to define the influence of genetic modifiers on manifestation of skeletal patterning phenotypes in Hoxb6 mutants by analyzing the mutation on the FVB genetic background; and (3) to characterize the expression of genes that are known mediators of skeletal patterning for their function in pathogenesis of Hoxb6 mutant phenotypes. The identification of modifier loci that act on skeletal patterning will reveal new molecular mechanisms in skeletal development and has important implications for the evolution of vertebrate body plans.